2 pathway

Journal Pre-proof Farrerol alleviates high glucose-induced renal mesangial cell injury through the ROS/ Nox4/ERK1/2 pathway Zhao Chen, Heyan Gao, Li Wang, Xiaotao Ma, Lifang Tian, Weihao Zhao, Ke Li, Yani Zhang, Fangxia Ma, Jiamei Lu, Lining Jia, Yanyan Yang, Rongguo Fu PII:

S0009-2797(19)31182-2

DOI:

https://doi.org/10.1016/j.cbi.2019.108921

Reference:

CBI 108921

To appear in:

Chemico-Biological Interactions

Received Date: 10 July 2019 Revised Date:

29 November 2019

Accepted Date: 10 December 2019

Please cite this article as: Z. Chen, H. Gao, L. Wang, X. Ma, L. Tian, W. Zhao, K. Li, Y. Zhang, F. Ma, J. Lu, L. Jia, Y. Yang, R. Fu, Farrerol alleviates high glucose-induced renal mesangial cell injury through the ROS/Nox4/ERK1/2 pathway, Chemico-Biological Interactions (2020), doi: https://doi.org/10.1016/ j.cbi.2019.108921. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Tittle: Farrerol alleviates high glucose-induced renal mesangial cell injury through the ROS/Nox4/ERK1/2 pathway Zhao Chen: Conceptualization, Methodology, Investigation, Writing original draft Heyan Gao: Methodology, Investigation, Software, Validation Li Wang, Xiaotao Ma, Lifang Tian: Methodology, Investigation, Formal analysis Weihao Zhao, Ke Li, Yani Zhang, Fangxia Ma, Jiamei Lu: Investigation, Resources Lining Jia: Software, Data Curation Yanyan Yang: Writing original draft, Visualization Rongguo Fu: Conceptualization, Writing Review&Editing, Supervision

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Farrerol alleviates high glucose-induced renal mesangial cell injury through the

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ROS/Nox4/ERK1/2 pathway

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Zhao Chen1,&, Heyan Gao2,&, Li Wang1, Xiaotao Ma1, Lifang Tian1, Weihao Zhao1, Ke Li1, Yani

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Zhang1, Fangxia Ma1, Jiamei Lu1, Lining Jia1, Yanyan Yang1, Rongguo Fu1,*

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1

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Shaanxi, China

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&

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*corresponding author: Rongguo Fu

Department of Nephrology, the Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an,

Department of Nephrology, the First People's Hospital of XianYang, Xian Yang, Shaanxi, China Zhao Chen and Heyan Gao contribute equally to this work.

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The Second Affiliated Hospital of Xi’an Jiaotong University, 710004, No. 157 Xiwu Road,

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Xincheng District, Xi’an, Shaanxi, China

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E-mail address: [email protected]

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1

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Abstract

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Hyperproliferation and oxidative stress induced by hyperglycemia in mesangial cells plays

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crucial roles in the pathological process of diabetic nephropathy. Farrerol, isolated from

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rhododendron leaves, possesses broad anti-oxidative and anti-inflammatory properties towards

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several diseases, but its role in diabetic neuropathy remains unclear. The aim of this study was to

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evaluate the effects of farrerol in high glucose induced mesangial cell injury, and to explore

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underlying molecular mechanisms. Our results showed that high glucose in vitro conditions

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significantly stimulated cell proliferation, inflammatory cytokine secretion, extracellular matrix

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deposition, excessive oxidative stress, and NADPH oxidase activity in mesangial cells. Levels of

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NADPH oxidase 4 (Nox4) expression, ERK1/2 phosphorylation, and TGF-β1/Smad2 activation

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were significantly induced by high glucose conditions in mesangial cells. Inversely, farrerol

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treatments at 40, 60, and 80 µM concentrations, dose-dependently alleviated this molecular

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damage by high glucose in mesangial cells. We also found that restoration of Nox4 expression

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abolished the protective effects of farrerol on high glucose-induced proliferation and reactive

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oxygen species generation. Furthermore, pretreatment with the Nox4 inhibitor diphenyliodonium

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or the ERK1/2 pathway inhibitor PD98059, displayed similar ameliorated effects of farrerol on

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high glucose-induced mesangial cell damage. Taken together, these data suggest that farrerol

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displays protective effects on high glucose induced mesangial cell injury, partly through the Nox4-

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mediated ROS/ERK1/2 signaling pathway. These observations may provide novel insights into the

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application of farrerol as a diabetic neuropathy treatment.

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Keywords: Diabetic neuropathy; Mesangial cells; Farrerol; High glucose; Nox4; ROS

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2

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1. Introduction

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Diabetic nephropathy (DN), as the most common and prevalent complication of diabetes

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mellitus (DM), significantly contributes to end-stage renal failure [1]. Though the exact

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pathogenesis of DN remains unclear, increasing evidence has demonstrated that the expansion of

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the glomerular mesangium, glomerular hypertrophy, and the thickness of the glomerular basement

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membranes are involved in DN pathophysiological changes [2]. Mesangial cells (MCs) are located

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around glomerular capillaries of the kidneys, and perform crucial roles in balancing renal

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functions under physiological conditions, whereas under pathological conditions, excessive

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reactive oxygen species (ROS) induced by chronic hyperglycemia exposure, potentially activates

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glucose downstream signaling cascades, directly stimulating abnormal cell proliferation,

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extracellular matrix (ECM) deposition and MC inflammation, ultimately leading to diabetic

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glomerulosclerosis and renal fibrosis [3]. Therefore, the elucidation of underlying mechanisms in

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MC mediated hyperglycemia injury could provide valuable insights into understanding DN

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pathogenesis.

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The nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Nox) family

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(including Nox 1–5 and Duox 1–2) functions as major ROS producers in response to diverse

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stimuli in nonphagocytic cells [4]. Nox4 as a key Nox isoform is characterized by the production

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of hydrogen peroxide (H2O2) under pathological conditions, and is highly expressed in the kidney

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and particularly the mesangium [5]. Studies have shown that elevated Nox4 expression and Nox4-

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dependent ROS production account for hyperglycemia-induced diabetes, which is ameliorated by

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insulin administration [6]. Furthermore, Nox4-derived ROS has been shown to stimulate renal

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hypertrophy and fibronectin accumulation in diabetic glomeruli and cortex [7]. Moreover, chronic

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exposure to high glucose has been confirmed to directly contribute to the augmentation of Nox4

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expression in MCs [8]. Knockdown of Nox4 prevents angiotensin II induced mitochondrial

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superoxide production and renal fibrosis in glomerular MCs [9]. Accordingly, it is hypothetical to

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propose that suppression of Nox4-mediated oxidative stress in MCs could be used to interference

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DN progression.

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Farrerol (FAR) (C17H16O5, molecular weight, 300.31 Da, Fig. 1A) is a type of 2,3-dihydro-

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flavonoid isolated from rhododendron leaves [10]. Accumulating pharmacological evidence has

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shown that FAR elicits anti-inflammatory, antioxidant, antibacterial and anti-cancer activities 3

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through several signaling pathways. For instance, FAR has been reported to alleviate β-amyloid-

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induced oxidative stress and inflammation in a microglial cell line via the NF-E2-related factor

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(Nrf2) pathway [11]. Furthermore, FAR appears to alleviate aortic lesions via the enhancement of

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nitric oxide synthase (eNOS) and the reduction of NADPH oxidase activity in hypertensive rats

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[12]. However, whether FAR possesses protective effects towards DN and its underlying

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mechanisms still remains unclear. Therefore, in this study we sought to investigate FAR function

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on hyperglycemia-stimulated MCs and explore its molecular mechanisms.

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2 Materials and methods

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2.1 Cell culture and treatment

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Rat MCs were obtained from the American Type Culture Collection (ATCC, Manassas, USA)

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and cultured in Dulbecco’s modified eagle’s medium (DMEM, Hyclone, UT, USA), supplemented

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with 10% fetal bovine serum (FBS, Gibco, NY, USA) and 1% penicillin/streptomycin (Hyclone)

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at 37°C under 5% CO2 humidified conditions.

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FAR (Sigma-Aldrich, MO, USA) was dissolved in dimethyl sulfoxide (DMSO, Sigma-

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Aldrich) to a stock concentration of 100 mg/ml and freshly diluted into DMEM at concentrations

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of 5, 10, 20, 40, 60, and 80 µM. Concentrations were supplemented to MCs under normal glucose

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(NG, 5.5 mM) or high glucose (HG, 30 mM) culture conditions for 48 h. To modulate the ROS-

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Nox4-ERK1/2 pathway, MCs were pretreated with the ERK inhibitor PD98059 (10 µM), the

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Smad2 inhibitor LY2109761 (10 µM) or the Nox4 inhibitor diphenyliodonium (DPI, 10 µM) for 1

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h, respectively and then transiently transfected with the pcDNA3.1-Nox4 overexpressing plasmid

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(Nox4) and its empty vector (GenePharma, Shanghai, China), using lipofectamine 2000

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(Invitrogen, Carlsbad, CA, USA). After this, MCs were exposed to HG (30 mM) and FAR (60

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µM) for 48 h.

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2.2 Cell viability assay

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The MTT assay was performed to assess FAR effects on cell viability. MCs were seeded in

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96-well plates (5×103 cells/well) overnight, and then incubated under NG or HG conditions with

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various FAR concentrations. After 48 h incubation, 10 µl MTT (5 mg/ml) was added to each well

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and incubated for a further 4 h. After this period, the medium was discarded and 150 µl DMSO

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was added to each well to dissolve the precipitate. The optical density (OD490nm) was determined

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on a microplate reader (BioTek, VT, USA). 4

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2.3 Measurement of intracellular ROS production

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Intracellular ROS levels were evaluated by the dichloro-dihydro-fluorescein diacetate

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(DCFH-DA) (ROS assay kit, Beyotime, Shanghai, China) method. Briefly, MCs were pretreated

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with FAR for 48 h under HG conditions and then incubated with 10 µmol/l DCHFH-DA for 20

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min at 37℃ in the dark according to the manufacturer’s instructions. After rinsing, fluorescence

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was observed under a fluorescence microscope (Leica, Germany) and fluorescence intensity was

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determined using a microplate reader (BioTek) at an excitation wavelength of 488 nm and an

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emission wavelength of 525 nm.

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2.4 Detection of MDA and SOD activity

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MDA levels were determined by the lipid peroxidation MDA assay kit (Beyotime) following

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the manufacturer’s instructions. In brief, MCs were lysed and centrifuged at 12,000×g for 10 min

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at 4°C, and the resulting supernatant was mixed with 0.37% thiobarbituric acid (TBA) and

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incubated at 100°C for 15 min. Next, the mixture was centrifuged at 1000×g for 10 min and the

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supernatant was collected for absorbance measurements at 523 nm on a microplate reader

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(BioTek). SOD activity was measured using the total superoxide dismutase assay kit and the

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WST-8 enzyme (Beyotime) according to manufacturer’s instructions. MCs were lysed and

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incubated with the WST-8 enzyme working solution at 37°C for 30 min. SOD activity was

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determined at 450 nm on a microplate reader (BioTek).

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2.5 NADPH oxidase activity assay

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NADPH oxidase activity was determined using the amplite colorimetric NADPH assay kit

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(AAT Bioquest, CA, USA) according to manufacturer’s instructions. In brief, MCs were harvested

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and centrifuged at 800×g for 2 min. The supernatant was mixed with the NADPH working

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solution and incubated at room temperature for 2 h. The absorbance was monitored at 460 nm on a

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microplate reader (BioTek).

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2.6 ELISA assay

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Inflammatory cytokine levels, including the tumor necrosis factor-α (TNF-α), interleukin

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(IL)-6 and IL-1β in MC supernatants were quantified using commercial ELISA kits (R&D

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systems, MN, USA) following manufacturer’s instructions. The absorbance was measured at 450

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nm on a microplate reader (BioTek).

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2.7 qRT-PCR analysis 5

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Total RNA was isolated from MCs using Trizol reagent (Tiangen Biotech, Beijing, China).

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Equal RNA concentrations of each sample were reversely transcribed into cDNAs using the

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TaqMan reverse transcription kit (Takara, Dalian, China) according to manufacturer’s instructions.

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The qPCR was performed using a SYBR-Green PCR master mix (Applied Biosystems, CA, USA)

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on an ABI 7500 Real-Time PCR system (Applied Biosystems). Experimental conditions were: 40

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cycles of denaturation at 95°C for 15 s, annealing at 55℃ for 35 s and extension at 72℃ for 1

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min. The relative expression of each gene was assessed by the 2-

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GAPDH levels. GAPDH was used as an internal control. The specific primers were listed as

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follows: GAPDH, forward: 5’- TGG ATA GGG TGG CCG AAG TA-3’ and reverse: 5’-GGA

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AAC CCT GCC ATC CAT CA-3’; IL-6, forward: 5’- CTC TTC TCT GGT TGC CCC T-3’ and

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reverse: 5’- CGA CTC CTT GCC TTC TAC CC-3’; IL-1β, forward: 5’- TGG ACG TGC AAT

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AAC TGC CT-3’ and reverse: 5’- CCG TGT GGT ATT GGT GGG AA-3’; TNF-α, forward: 5’-

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CTC GAG TGA CAA GCC CG TAG -3’ and reverse: 5’- CCC ACA CTT CAC TTC CGG TT-3’;

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fibronection, forward: 5’- AAC ACA AGA TCA GCA CCC CC -3’ and reverse: 5’- AAT GTG

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CGA GCC ACT TAC CT-3’; laminin, forward: 5’- TTG GAA ATA ACG CCG TGG GA -3’ and

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reverse: 5’- TCT GTT CCC AAG TCG AAG CC -3’; collagen IV, forward: 5’- CCT CAT GTC

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AAG CCA AGG GT -3’ and reverse: 5’- TGA TGG ATG GCT GCT CGT TT -3’; MMP-2

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forward: 5’- AAA TTC GGG GCT GTG CTG TA-3’ and reverse: 5’- AAA GGC GGA GTT ACA

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AGG GG -3’ and MMP-9, forward: 5’- CAG CGA GAC ACT AAA GGC CA-3’ and reverse: 5’-

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AAT GGA TCC GCT CGG TCT TC-3’; Nox2, forward: 5’- GGA TGA AGA CTT GCT GCC CT

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-3’ and reverse: 5’- GCT CCC TAT GGC ATT CCC TC -3’; Nox4, forward: 5’- CTG GTG TTG

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TTG TCA GGG GT -3’ and reverse: 5’- ACT GTG ACT GTA AGA GCG CC -3’; p22phox,

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forward: 5’- AGG GAT TCT GAG TGC GGT TG -3’ and reverse: 5’- GGC TTG GGT GGA

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CGA TGT TA -3’.

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2.8 Western blot analysis

△△Ct

method and normalized to

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MCs were harvested and lysed with RIPA lysis buffer (Beyotime). The protein concentration

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was measured by the BCA assay kit (Beyotime) according to manufacturer’s instructions.

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Subsequently, 30 µg protein from each sample was separated on a 12% SDS-PAGE gel and

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transferred to a PVDF membrane (Millipore, MA, USA). After blocking with 5% non-fat milk for

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1 h at room temperature, the membranes were incubated with specific primary antibodies against 6

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Nox4, Nox2, p22phox, fibronectin, laminin, collagen IV, MMP-2, MMP-9, TGF-β, Smad2,

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phosphorylation (p)- Smad2, Smad-4, ERK1/2, p-ERK1/2 and GAPDH (Abcam, Cambridge, MA,

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USA) overnight at 4°C, and then incubated with appropriate horseradish peroxidase (HRP)-

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conjugated secondary antibodies (Santa Cruz Biotechnology, CA, USA) at 37°C for 2 h. Protein

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bands were detected using the chemiluminescence (ECL) detection system (Bio-Rad, CA, USA)

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and band intensities were calculated using ImageJ software (NIH, MD, USA). GAPDH was used

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as an internal control.

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2.9 Statistical analysis

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Data were presented as the mean ± standard deviation (SD) using GraphPad Prism software

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(GraphPad, San Diego, CA, USA). Each experiment was independently repeated at least three

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times. Comparisons between two groups were analyzed using unpaired Student’s t-test and

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multiple comparisons were assessed using one-way analysis of variance (ANOVA) followed by

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Bonferroni adjustments. Statistical significance was observed as P < 0.05.

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3. Results

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3.1 The inhibitory effects of FAR on HG-induced MC viability

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The MTT assay was used to assess the cytotoxic effects of FAR on MC viability. Our results

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showed that when MCs were treated with FAR at the concentrations of 5, 10, 20, 40, and 60 µM

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but not 80µM showed no cytotoxicity on MCs under NG conditions (P > 0.05, Fig. 1B). In

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addition, the effect of FAR on HG-stimulated cell viability was also assessed in MCs. The MTT

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assay revealed that MC viability was significantly increased under HG conditions, whereas FAR at

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20, 40, 60 and 80 µM concentrations reduced cell viability induced by HG treatments (Fig. 1C).

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Hence, FAR at the 20, 40, and 60 µM concentrations were chosen for the following study.

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3.2 The inhibitory effects of FAR on HG-induced inflammatory responses and ECM

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formation in MCs

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The effects of FAR on inflammatory cytokines (IL-6, IL-1β, and TNF-α) in MCs were

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assessed by ELISA (Fig. 2A). Levels of IL-6, IL-1β, and TNF-α in MC supernatants were

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significantly enhanced under HG stimulation (P < 0.01), but were inhibited by dose-dependent

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FAR treatments at 20, 40, and 60 µM. Moreover, qRT-PCR revealed that increased mRNA

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expression levels of IL-6, IL-1β, and TNF-α in MCs induced by HG were significantly alleviated

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by FAR co-treatments (Fig. 2B). Next, the effects of FAR on ECM deposition in HG-induced MC 7

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damage were assessed. Western blot assays showed that HG stimulation significantly increased

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fibronectin, laminin and collagen IV protein levels, but markedly decreased MMP-2 and MMP-9

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protein levels in MCs when compared to the control group (P < 0.01, Fig. 3A). However, FAR

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treatments appeared to abolish these changes in MCs (Fig. 3A), and also, qRT-PCR data showed

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similar results (Fig. 3B). These results therefore indicate that FAR inhibits HG-induced

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inflammation and ECM accumulation in MCs.

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3.3 The antioxidant effects of FAR on HG-stimulated oxidative stress in MCs

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The antioxidant effects of FAR on ROS production, MDA levels and SOD activities were

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determined in MCs (Fig. 4A, B, and C). HG-stimulated MCs showed a significant elevation in

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ROS production and MDA levels when compared to the control group (P < 0.01). However, this

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HG-induced ROS and MDA production in MCs was dose-dependently abrogated by the co-

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treatment of MCs with 20, 40, and 60 µM FAR concentrations (P < 0.05, Fig. 4A and B).

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Furthermore, SOD activity was suppressed in HG treated MCs (P < 0.05, Fig. 4C), but was

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reversely attenuated by FAR co-treatment in a dose-dependent manner.

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3.4 The effects of FAR on HG-induced NADPH oxidase activation in MCs

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Next, we investigated NADPH oxidase activity in MCs, due to its crucial role in intracellular

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ROS generation. As shown in Fig. 5A, HG treatment significantly enhanced NADPH oxidase

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activity in MCs when compared to the control group (P < 0.05), whereas co-treatment of MCs

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with FAR apparently attenuated HG-induced NADPH oxidase activity in a FAR dose-dependent

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manner (P < 0.05). Accordingly, we assessed the expression levels of Nox2 and Nox4, two key

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Nox isoforms of NADPH oxidase in MCs, by qRT-PCR and western blot assay. We found that HG

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treatment of MCs resulted in a significant enhancement of Nox2 and Nox4 expression, at the

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mRNA and protein levels (P < 0.01, Fig. 5B and C). However, treatment with FAR, in a dose-

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dependent manner, reduced Nox4 expression but did not affect Nox2 expression in HG-stimulated

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MCs. The Nox4 subunit, p22phox was also detected in MCs under HG conditions, but we

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observed that the HG-induced elevation of p22phox was gradually reduced by FAR treatments in a

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dose-dependent manner. Therefore, these data indicate that Nox4 may be involved in FAR-

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mediated anti-oxidative stress in HG-treated MCs.

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3.5 Restoration of Nox4 suppresses the protective effects of FAR on HG-induced MC damage

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To determine whether Nox4 was the potential target for FAR in exerting antioxidant effects in 8

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HG-treated MCs, Nox4 expression was restored in MCs by transfecting the Nox4 overexpressing

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plasmid into cells. As shown (Fig. 6A), Nox4 expression levels were significantly increased by the

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Nox4 overexpression plasmid in HG-stimulated MCs (P < 0.01), whereas, Nox4 inhibitor

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diphenyliodonium (DPI) suppressed Nox4 expression in HG-treated MCs (P < 0.01).

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Furthermore, the reduced expression of Nox4 induced by FAR (60 µM) or DPI treatment was

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reversed with the Nox4 overexpressing plasmid under HG conditions when compared to empty

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vector transfection MCs (P < 0.01). We also explored the effects of FAR on HG-induced MC

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proliferation and oxidative stress with Nox4 overexpression. Compared with the HG+FAR group,

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exogenous expression of Nox4 significantly increased cell growth in the FAR or DPI co-treated

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group (Fig. 6B and C, P < 0.01). Furthermore, Nox4 up-regulation reversely recovered the

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decreased NADPH oxidase activation and ROS production induced by FAR or DPI treatment (P <

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0.01). These findings indicate that FAR elicits protective effects in MCs under HG stimulation,

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through the inhibition of Nox4-mediated ROS production.

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3.6 FAR inhibits HG-induced ERK1/2 and TGF-β activation in MCs

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To explore putative FAR-mediated signaling cascades under HG-induced MC damage, the

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expression of components from the ERK1/2 and TGF-β1/Smad2 signaling pathways were

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assessed by western blotting. The results showed that TGF-β, phosphorylation (p)- Smad2, Smad-

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4, and p-ERK1/2 expression levels were significantly increased by HG stimulation in MCs, but

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such increases were inhibited by FAR treatments in a dose-dependent manner (Fig. 7A).

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Moreover, pretreatment with DPI also reduced the up-regulation of p-ERK1/2, TGF-β, p- Smad2,

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and Smad-4 activation induced by HG.

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To further verify that the ERK1/2 and TGF-β1/Smad2 pathways participate in the FAR-

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mediated protection of HG-stimulated MCs, the ERK inhibitor, PD98059 (10 µM) and the Smad2

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inhibitor, LY2109761 (10 µM) were used to inhibit ERK1/2 and TGF-β1/Smad2 pathway

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activities (Fig. 7B). We found that, similar to the FAR (60 µM) and DPI (10 µM) treatment data,

241

pretreatment with PD98059 significantly abolished HG-induced up-regulation of p-ERK1/2, TGF-

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β, p- Smad2 and Smad-4 (P < 0.05). Furthermore, the HG-induced expressions of TGF-β, p-

243

Smad2 and Smad-4 were reversed with LY2109761 inhibitor incubation, in contrast, LY2109761

244

failed to suppress p-ERK1/2 expression in HG-stimulated MCs. Interestingly, FAR, DPI and

245

PD98059 repressed HG-induced Nox4 expression, whereas, no changes in Nox4 levels was found 9

246

in MCs under LY2109761 treatment (Fig. 7B). These findings indicate that Nox4 and ERK1/2

247

pathways form a signaling axis to regulate the downstream TGF-β1/Smad2 pathway in MCs,

248

under HG conditions.

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3.7 The suppression of the ERK/TGF-β axis reverses HG-induced injury in MCs

250

To determine whether the ERK and TGF-β1/Smad2 axis participated in HG-induced damage

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in MCs, cells were pretreated with PD98059, LY2109761 and DPI for 1h. The results showed that

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the increased cell proliferation induced by HG in MCs was markedly inhibited by PD98059,

253

LY2109761 and DPI treatments (Fig. 8A). Furthermore, PD98059 and DPI, but not LY2109761

254

abrogated the stimulated ROS levels in MCs under HG conditions (Fig. 8B). The elevated mRNA

255

levels of fibronectin, laminin and collagen IV induced by HG, were apparently attenuated in the

256

presence of PD98059, LY2109761 and DPI inhibitors (Fig. 8C). Taken together, these results

257

suggest that the ERK and TGF-β1/Smad2 signaling pathways are involved in HG-induced cell

258

injury in MCs.

259

4. Discussion

260

The early stages of DN are characterized by the increased proliferation of MCs and abundant

261

ECM deposition, which often results in hyperplasia of the glomerular mesangium, renal fibrosis,

262

and end-stage renal damage [13]. Therefore, MC dysregulation has been implicated as a

263

prominent event in the development of renal injury, under hyperglycemic conditions [14]. In this

264

study, an in vitro DN model was established using rat MCs, subjected to high glucose stimulation.

265

FAR, extracted from rhododendron leaves, is a new isolate of the 2,3-dihydro-flavonoids. It has

266

been reported that FAR inhibits the fetal bovine serum-induced abnormal proliferation of rat

267

thoracic aorta vascular smooth muscle cells via its interaction with estrogen receptors [15].

268

Previous studies have also revealed that FAR inhibits gastric carcinoma cell growth by inducing

269

G0/G1 phase cell-cycle arrest, and suppressing human umbilical vein endothelial cell proliferation

270

through the induction of apoptosis [16]. Consistent with previous findings, in this study, FAR

271

treatments effectively suppressed HG-induced MC hyperproliferation in a dose-dependent manner,

272

thereby prompting us to explore the underlying molecular mechanisms.

273

Recently, accumulating evidence has suggested that inflammation and ECM accumulation

274

contributes to kidney damage in hyperglycemia [17]. Chronic inflammatory responses induced by

275

HG stimulation in MCs could result in MC over-proliferation and expansion through the 10

276

production of excessive ECM [18]. Fibronectin, laminin and collagen IV as the prominent

277

components of the mesangial matrix are abundantly synthesized under hyperglycemic stimulation

278

[19]. In contrast, matrix metalloproteinase (MMPs) are iron-dependent enzymes that induce

279

matrix degradation of the ECM, and have been shown to be downregulated by HG in MCs [20]. In

280

this study, in conjunction with the increased production of inflammatory cytokines, including IL-

281

6, IL-1β, and TNF-α induced by HG treatment, we observed enhanced expression of fibronectin,

282

laminin, collagen IV and reduced expression of MMP-2 and MMP-9 in HG-stimulated MCs,

283

however, FAR treatments dose-dependently abrogated this abnormal inflammation and ECM

284

deposition in MCs. Similarly, previous studies have demonstrated the anti-inflammatory effects of

285

FAR on other cell models, such as IL-1β-stimulated human osteoarthritic chondrocytes [21], LPS-

286

induced human gingival fibroblasts [22] and MPP+-induced microglia cells [23]. Accordingly, our

287

findings demonstrated that FAR possessed the properties to suppress HG-induced inflammation

288

and ECM accumulation in MCs.

289

Until now, the clinical antioxidant agents used for DN therapy were always accompanied by

290

adverse cardiovascular complications, implying an urgent need to seek alternative DN

291

antioxidants [24]. The progression and development of DN is closely associated with the

292

excessive generation of oxidative stress induced by hyperglycemia in the kidney, which is

293

involved in inflammation and ECM accumulation in glomerular MCs [25]. HG-induced ROS

294

accumulation interrupts the balance between pro- and anti-oxidants, which in turn breaks

295

antioxidant defense systems, causing tissue degeneration, DNA and protein damage and lipid

296

peroxidation [17]. Under HG conditions, the reduced activities of antioxidant enzymes such as

297

SOD, CAT, and GSH means they fail to scavenge oxygen free radicals and alleviate ROS-

298

mediated oxidative damage. In addition, lipid peroxidation products, such as MDA, triggered by

299

free radicals directly reflect cell damage in MCs [26]. In this study, we observed increased ROS

300

generation in MCs under HG stimulation, and exposure to HG elevated MDA activity and

301

decreased SOD activity in MCs. However, FAR treatment dose-dependently ameliorated HG-

302

induced ROS damage, suggesting a potential antioxidant role of FAR on HG-induced oxidative

303

stress in MCs.

304

Growing evidence has suggested that the major source of ROS production, induced by HG in

305

MCs is attributed to the NADPH oxidases (Noxs), which are the major isoforms of the NADPH 11

306

oxidases, Nox2 and Nox4 that are predominantly expressed in MCs under HG stimulation [27,

307

28]. In our study, similar elevations of Nox2 and Nox4 were observed in MCs exposed to HG.

308

Previous studies have proposed that various drugs exert their antioxidant properties via Nox

309

modulation in DN pathology. For example, a recent study showed that the suppression of Nox

310

oxidase could markedly block diabetes-induced ROS and ECM expansion in MCs in diabetic

311

animals [29]. Accordingly, we investigated whether FAR exerted a regulatory role on Noxs in HG-

312

induced MC injury. Our results revealed that FAR treatment significantly downregulated Nox4,

313

but not Nox2 in a concentration-dependent manner, suggesting that Nox4 is the glomerular

314

antioxidant target of FAR. Moreover, we also found that FAR ameliorated the expression of

315

p22phox, which serves as the membrane-associated subunit interacting with Nox4, and is closely

316

associated with Nox4-based NADPH oxidase in MCs in response to HG stimulation [30].

317

Furthermore, we confirmed that under HG conditions, the alleviated effects of FAR or the Nox4

318

inhibitor DPI on cell hyperproliferation and ROS production in MCs induced by HG, could be

319

abrogated by the restoration of Nox4 expression, implying the involvement of Nox4 in FAR-

320

mediated relief of HG-induced ROS damage. Admittedly, though DPI is widely used as a general

321

flavoprotein to inhibit NADPH oxidase activity, its non-specific inhibitory properties have also

322

been noted against xanthine oxidase, eNOS and proteins of the mitochondrial electron transport

323

chain [31]. Therefore, the use of DPI on NADPH oxidase-mediated effects may be overestimated,

324

suggesting more specific inhibitors are required in the future research.

325

As a member of the MAPK family, the ERK1/2 pathway could be activated by

326

hyperglycemia in the diabetic kidney and contributes to DN progression. It has been shown that

327

the activation of the ERK1/2 pathway, induced by HG, is mediated by ROS accumulation and is

328

required for ECM accumulation in MCs [32]. Furthermore, a previous study has illustrated that the

329

inhibition of HG-induced EKR1/2 activation, results in the attenuated oxidative stress and cell

330

proliferation of MCs [33]. In our study, we observed that the phosphorylation of ERK1/2 induced

331

by HG was dose-dependently suppressed by FAR treatment. Moreover, DPI, the Nox4 inhibitor

332

significantly abrogated HG-induced ERK1/2 activation, which was consistent with a previous

333

study [34]. Interestingly, when compared to FAR effectiveness, the suppression of the ERK1/2

334

pathway by the ERK inhibitor, PD98059 also showed an inhibitory effect on HG-induced Nox4

335

expression, as well as an alleviating effect on HG-induced ROS generation, implying that cross12

336

talk or a parallel regulatory mechanism may exist between Nox4-mediated ROS and ERK1/2

337

activation in HG-induced MC injury. Furthermore, under oxidative stress induced by HG,

338

activated TGF-β/Smad2 signaling is responsible for the proliferation and ECM deposition of MCs,

339

and plays prominent roles in the development of early DN [35]. It has been demonstrated that

340

once TGF-β1 is activated by diabetic stimuli such as HG, its downstream receptor Smad2

341

becomes activated and interacts with Smad4 to facilitate its translocation into the nucleus to

342

promote ECM accumulation and inflammation [36]. In our study, similar to the effects of FAR and

343

DPI, PD98059 also displayed an inhibitory effect on TGF-β1/Smad2 activation in HG-induced

344

MC damage. Nevertheless, though pre-treatment with the TGF-β1/Smad2 pathway inhibitor

345

LY2109761 showed attenuated effects on HG-induced MC proliferation and ECM deposition, it

346

had no effect on ROS generation, Nox4 expression or ERK1/2 activation. Taken together, these

347

data suggest that the TGF-β1/Smad2 pathway may act as one of the downstream ERK1/2

348

pathways involved in FAR-mediated antioxidative effects on HG-induced MC injury.

349

In conclusion, this study demonstrated that hyperglycemia stimulated hyperproliferation,

350

inflammation, ROS generation, ECM accumulation, ERK1/2 and TGF-β1/Smad2 activation in

351

MCs. Treatment with FAR dose-dependently suppressed HG-induced MC damage through the

352

Nox4/ROS/ERK/TGF-β signaling pathway (Fig. 9). These data provide a possible mechanism

353

whereby FAR alleviates ECM deposition and oxidative stress of MCs induced by HG. It has been

354

widely confirmed that numerous signaling pathways contemporarily participate in oxidative stress,

355

suggesting their inter-relationships are very complicated. For example, a previous study has

356

theorized that the antioxidative potential of FAR in RAW 264.7 cells is through Nrf2 mediated

357

Akt, p38 and ERK signaling [37]. Therefore, further in-depth investigations, similar to this one,

358

are required to explore and determine the roles of other signaling pathways involved in FAR-

359

mediated antioxidative mechanisms in HG-induced MC damage.

360 361

Acknowledgements

362

This study was supported by grants from the “Science & Technology Research and Development

363

Program of Shaanxi Province (No. 2016SF-176)”.

364

Conflict of interest

365

There is no conflict of interest. 13

366

Reference

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466

Figure legends

467

Fig. 1. The inhibitory effects of FAR on HG-induced MC viability. (A) The molecular structure of

468

FAR (C17H16O5, molecular weight; 300.31 Da). The effects of FAR (5, 10, 20, 40, 60, and 80 µM)

469

on MC viability under NG (B) and HG (C) conditions over a 48 h incubation were determined by

470

MTT assay. NG, normal glucose; HG, high glucose. Data were presented as the mean ± standard

471

deviation (SD) and all assays were performed in triplicate. *P < 0.05, **P < 0.01.

472

Fig. 2. The inhibitory effects of FAR on HG-induced inflammatory responses in MCs. The effects

473

of FAR on IL-6, IL-1β, and TNF-α levels in HG-induced MC damage were quantified by ELISA

474

(A) and qRT-PCR (B). Data were presented as the mean ± SD and all assays were performed in

475

triplicate. *P < 0.05, **P < 0.01.

476

Fig. 3. FAR suppresses HG-induced ECM accumulation in MCs. Protein and mRNA expression of

477

fibronectin, laminin, collagen IV, MMP-2 and MMP-9 in MCs under different treatment of FAR

478

were assessed by western blot (A) and qRT-PCR (B). Data were presented as the mean ± SD and

479

assays were performed in triplicate. *P < 0.05, **P < 0.01.

480

Fig. 4. The antioxidant effects of FAR on HG-stimulated oxidative stress generation in MCs. (A)

481

Representative images of ROS production in HG-induced MC damage with FAR treatments (20,

482

40, and 60 µM) for 48 h as determined by the DCFH-DA fluorescent probe. (B) The relative

483

fluorescence intensity of ROS levels in HG-induced MC damage with FAR treatments (20, 40,

484

and 60 µM) for 48 h, was measured by microplate reader. MDA levels (C) and SOD activity (D)

485

were detected in HG-induced MC damage with FAR treatments (20, 40, and 60 µM) for 48 h.

486

Data were presented as the mean ± SD and assays were performed in triplicate. *P < 0.05, **P <

487

0.01.

488

Fig. 5. The effects of FAR on HG-induced NADPH oxidase activation in MCs. (A) NADPH

489

oxidase activity was measured in HG-induced MC damage, with FAR treatments (20, 40, and 60

490

µM) for 48 h. mRNA and protein expression of Nox2, Nox4, and p22phox were assessed by qRT-

491

PCR (B) and western blot (C) in HG-induced MC damage, with FAR treatments (20, 40, and 60

492

µM) for 48 h. Data were presented as the mean ± SD and assays were performed in triplicate. *P <

493

0.05, **P < 0.01.

494

Fig. 6. The restoration of Nox4 suppresses the protective effects of FAR on HG-induced MC

495

damage. (A) The expression of Nox4 using the pcDNA3.1-Nox4 overexpressing plasmid or DPI 17

496

(10 µM) pretreatment in HG-induced MC damage in the absence or presence of FAR (60 µM) was

497

assessed by western blot. (B) Cell proliferation of HG-induced MC damage, plus Nox4

498

transfection, FAR (60 µM), or DPI (10 µM) co-treatment for 48h was detected by MTT assay.

499

Changes in NADPH oxidase activation (C) and ROS levels (D) in HG-induced MC damage, plus

500

Nox4 transfection, FAR (60 µM), or DPI (10 µM) co-treatment for 48h were measured. Data were

501

presented as the mean ± SD and assays were performed in triplicate. *P < 0.05, **P < 0.01.

502

Fig. 7. FAR inhibits HG-induced ERK1/2 and TGF-β1/Smad2 pathway activation in MCs. (A)

503

FAR treatment increased the activation of ERK1/2 and TGF-β1/Smad2 signaling pathways as

504

detected by western blot. Cells were pretreated with DPI (10 µM) and exposed to HG and FAR

505

(20, 40, and 60 µM) for 48 h. (B) The effects of the ERK inhibitor PD98059 (10 µM) and the

506

Smad2 inhibitor LY2109761 (10 µM) on Nox4, p-ERK1/2, TGF-β, p- Smad2, and Smad-4

507

expression levels were examined by western blot. Cells were pretreated with the ERK inhibitor

508

PD98059 (10 µM), the Smad2 inhibitor LY2109761 (10 µM) and DPI (10 µM) for 1 h and

509

exposed to HG and FAR (60 µM) for 48 h. Data were presented as the mean ± SD and assays were

510

performed in triplicate. *P < 0.05, **P < 0.01.

511

Fig. 8. Suppression of the ERK/TGF-β axis reverses HG-induced injury in MCs. (A) Cell

512

proliferation was determined by MTT assay. (B) The relative fluorescence intensity of ROS levels

513

was measured. (C) The mRNA expressions of fibronectin, laminin, and collagen I were measured

514

by qRT-PCR assay. Cells were pretreated with the ERK inhibitor PD98059 (10 µM), the Smad2

515

inhibitor LY2109761 (10 µM) and DPI (10 µM) for 1 h and exposed to HG for 48 h. Data were

516

presented as the mean ± SD and assays were performed in triplicate. *P < 0.05, **P < 0.01.

517

Fig.9. Schematic of FAR-mediated alleviation of HG-induced MC dysfunction via the

518

ROS/Nox4/ERK1/2 signaling pathway.

519

18

1. HG stimulated MC proliferation, inflammation, ECM deposition, and ROS production. 2. FAR dose-dependently inhibited HG-induced MC proliferation and ECM deposition. 3. FAR exerted anti-oxidative effect on HG-induced MCs via modulating ROS/Nox4/ERK. 4. TGF-β1/Smad2 was involved in FAR/Nox4-mediated ECM deposition in HG-induced MCs.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: